Curative therapy for autoimmune diseases will require reinstitution of immunological tolerance within those T and B cells that have lost the capacity to peacefully co-exist with self antigen. However, a lack of knowledge regarding the induction and maintenance of antigen-specific unresponsiveness (clonal anergy) hampers the development of such tolerance-inducing regimens. In the previous grant period, we reported that co- expression of Folate receptor-4 (FR4) and CD73 is strongly associated with the development of clonal anergy in pathogenic CD4+ T cells. Furthermore, we showed that the development and maintenance of anergy in these autoreactive CD4+ T cells was highly dependent on the activity of polyclonal Foxp3+ Tregs. We have now observed that the induction and maintenance of anergy is associated with the up-regulation of the lineage-directing nuclear factor C/EBP alpha during self antigen recognition. Furthermore, we have found that antigen-experienced polyclonal CD4+ T cells having a FR4hi CD73hi phenotype differentiate to Th1 and Tfh lineages that cause disease when transferred into lymphopenic hosts under conditions that prevent Foxp3+ Treg generation. Accordingly, the goals ofthe current proposal are to 1) determine the unique roles that Foxp3+ Tregs, yc cytokine receptors, TSC1, mTOR, and C/EBP alpha play in autoreactive CD4+ T cell clonal anergy reversal and differentiation to T effector/memory cell versus Foxp3+ T regulatory cell phenotypes, and 2) investigate the pathogenicity of a novel 'Anergic' CD44hi FR4hi CD73hi Foxp3- polyclonal CD4+ T cell subset, and determine the roles that C/EBPalpha and the Foxp3 CNS1 DNA sequence play in the differentiation of anergic self Ag-specific progenitor cells to either dangerous T effector/memory cell or protective Foxp3+ ITreg phenotypes. Ultimately, we intend to understand the effects of altered peripheral T cell homeostasis and deficient or dysfunctional Tregs on the reversal of anergy, to tip the balance back away from the differentiation of dangerous T effector/memory cells to the generation of ITregs capable of inducing and maintaining antigen-specific self tolerance.